CD40-binding activating antibodies

ABSTRACT

Disclosed are agonist anti-CD40 molecules, including monoclonal antibodies, which can bind to and stimulate professional and non-professional human antigen-presenting cells (“APCs”), enhance the stimulatory effect of CD40L on CD40 positive cells and/or induce phenotypical maturation of monocyte derived dendritic cells. Several such monoclonal antibodies are provided, and cell lines producing them have been deposited at the American Type Culture Collection.

RELATED APPLICATIONS

This application is a divisional patent application of U.S. patentapplication Ser. No. 09/773,866, filed on Feb. 1, 2001, now U.S. Pat.No. 7,172,759, which claims priority to U.S. Provisional ApplicationSer. No. 60/178,934, filed on Feb. 1, 2000, both of which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to a series of novel molecules and monoclonalantibodies that bind to and stimulate antigen presenting cells via theCD40 receptor expressed on such antigen presenting cells.

BACKGROUND OF THE INVENTION

Activation of the Immune System:

The immune system is capable of killing antilogous cells when theybecome infected by virus or when they transform into cancer cells. Sucha potentially dangerous mechanism is under tight control. When they havenot yet encountered their specific antigen, the immune system's T-killercells (CTL) circulate as inactive precursors. To be activated, theprecursor T-killer cell must recognize its specific antigen peptide,presented by MHC class I molecules on professional antigen presentingcells (APC). This antigen specific cellular interaction is, however, notenough to fully activate the CTL, notwithstanding the co-stimulatorysignals from the APC.

Until recently it was believed that a T-helper cell that recognises thesame antigen on the same APC as the CTL is needed to fully activate theCTL. Upon activation, the specific T-helper cell would supply cytokinessuch as IL-2 needed for the activation of the CTL. Guerder and Matzinger(J. Exp. Med. 176:553 (1992)), however, proposed the “licensing” modelfor CTL activation. In this model it was suggested that the T-helpercell, when recognising its antigen on a professional APC, would deliveran activation signal to the APC that as a result would be able tosubsequently activate a CTL without the need for the T-helper cell to bepresent. Recently, the molecular mechanism of the licensing model waselucidated. Schoenberger et al. (Nature 393:480 (1998)) described thecrucial role of the CD40L-CD40 pathway in the licensing model.Activation of the T-helper cell by the dendritic cell (DC) results inthe up-regulation of the CD40L, which subsequently provides the signalthat empowers the DC for CTL priming by triggering the CD40 molecule onthe DC.

DC circulate through and are resident in the body tissues and at sitesof antigen deposition or introduction. After taking up antigens, theymigrate to the draining lymph nodes where they present antigen to the Tcells. It is well known that a DC needs to be activated to performoptimally. Resting DC express only low levels of MHC and co-stimulatorymolecules and are poor stimulators of T cells. DC can be activated byinflammatory cytokines and bacterial products, which results inup-regulation of MHC and co-stimulatory molecules. Therefore, DC thathave encountered antigens under inflammatory conditions will readilyactivate T-helper cells when they arrive in the draining lymph nodes. Itis thus very likely that the combination of inflammatory cytokines atthe site of antigen uptake and the CD40L-CD40 interaction during theT-helper cell interaction result in an optimal capacity to license theDC for CTL activation.

The CD40 Molecule and the TNF Receptor Family:

The CD40 molecule belongs to the TNF receptor family of type Itransmembrane proteins. The members of this gene family (which includeamong others, the two receptors for TNF, the low-affinity nerve growthfactor receptor and the T cell activation antigen CD27, CD30, and CD95)are characterized by sequence homology in their cysteine-richextracellular domains (Armitage et al., Current Opinion in Immunology6:407 (1994)). The known ligands for the members of the TNF receptorfamily are homologous as well. Although TNF-α is a soluble cytokine, itis initially synthesized as a membrane associated molecule. Most of themembers of the TNF/CD40L receptor and the TNF/CD40 families are type IItrans-membrane proteins. These include: hTNF-α, hLT, hLT-β, hCD40L,hCD27L, hCD30L, cfECP1, myx VRh, mCD30, hCD27, hFas, m4-1BB, rOX-40,hTNFR-h, hTNFR-II, hTNFR-1 and hLNGFR. CD40 is best known for itsfunction in B-cell activation. The molecule is constitutively expressedon all B cells. CD40L-CD40 interaction can stimulate the proliferationof purified B cells and, in combination with cytokines, mediateimmunoglobulin production. Recent studies indicate that the distributionof the CD40 molecule is not as restricted as was originally postulated.Freshly isolated human monocytes express low levels of the CD40molecule, which can be upregulated by culturing them in the presence ofIFN-γ (Alderson et al., J. Exp. Med. 178:669 (1993)). Stimulation ofmonocytes via CD40 results in the secretion of pro-inflammatorycytokines such as IL-1 and TNF-α, toxic free radical intermediates suchas nitric oxide and up-regulation of the B7 co-stimulatory molecules.Human DC isolated from peripheral blood can also express the CD40molecule (Caux et al., J. Exp. Med. 180:263 (1994)). Ligation of CD40 onDC results in enhanced survival of these cells when cultured in vitro.As with monocytes, stimulation of DC via CD40 results in secretion ofpro-inflammatory cytokines such as IL-12 and TNF-α and up-regulation ofthe CD80/86 co-stimulatory molecules. In addition, it was recentlydemonstrated that activation of CD40 induces the capacity to stimulatethe activation of killer T cells (Schoenberger et al., Nature 393:480(1998)). Accordingly, activating CD40 by binding it with a ligand, suchas an antibody, would induce a number of humoral and cytotoxic effects,useful in inhibiting tumors.

SUMMARY OF THE INVENTION

The invention includes molecules able to bind to and activate CD40expressed on both professional and non-professional APCs. Theseagonistic molecules, following binding to a cell surface receptor,induce intracellular signal transduction, leading to the activation ofthe APCs expressing CD40. The molecules of the invention includemonoclonal antibodies, fragments thereof, peptides, oligonucleotides,and other chemical entities. Also included are peptides and genesinducing expression of anti-CD40 antibodies.

Such molecules can be used in combination, or in a bispecific ormultivalent form, including as bispecific antibodies, to cross-link CD40on the same cell, or to cross-link CD40 present on different cells.Either such cross-linking could cause a synergistic or additiveagonistic effect.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures and as described below, anti-CD40 antibodies produced byhybridoma clones generated by the methods of the invention are referredto as follows, in relation to the hybridomas deposited with the AmericanType Culture Collection (ATCC) and given the following ATCC depositaccession numbers: clone 4 (hybridoma MAb 186-4-1, ATCC Accession No.PTA-2996), clone 7 (hybridoma MAb 186-7-2, ATCC Accession No. PTA-2997),clone 15 (hybridoma MAb 186-15-1, ATCC Accession No. PTA-2998), clone 21(hybridoma MAb 186-21-1, ATCC Accession No. PTA-2993), clone 26(hybridoma MAb 186-26-3, ATCC Accession No. PTA-2999), clone 64(hybridoma MAb 186-64-1, ATCC Accession No. PTA-2994), clone 70(hybridoma MAb 186-70-3, ATCC Accession No. PTA-2995). The hybridomasproducing these antibodies were deposited with the ATCC (10801University Blvd., Manassas, Va. 20110-2209, USA) in accordance with theprovisions of the Budapest Treaty, on Jan. 31, 2001.

Applicants hereby state that all restrictions imposed by the depositoron the availability to the public of the deposited material will beirrevocably removed upon the granting of a U.S. patent based upon thisapplication.

FIG. 1 shows the induction of maturation of monocyte derived DC byanti-CD40 monoclonal antibodies. Monocyte derived immature dendriticcells were cultured for two days in the presence of anti-CD40 monoclonalantibodies or isotype matched control antibodies and then studied byFACS analysis for up-regulation of expression of CD80 anddown-regulation of the mannose receptor. Shown are the combined resultsof several experiments with the percentage of cells expressing CD83 inFIG. 1 a and the relative decrease in mean fluorescence intensity (MFI)of the mannose receptor expression in unstimulated cells (MFI arbitrarytaken as a value of 100) compared to stimulated cells in FIG. 1 b.

FIG. 2 shows induction of maturation of monocyte derived DC by anti-CD40monoclonal antibodies (clones 7, 15, 21, 48, 64 and 70). Monocytederived immature dendritic cells were cultured for two days in thepresence of anti-CD40 monoclonal antibody or isotype matched controlantibodies and then studied by FACS analysis for up-regulation ofexpression of CD80, CD83 and CD86 and down-regulation of expression ofthe mannose receptor. Data of one representative experiment are shown:the CD80 (FIG. 2 a), CD86 (FIG. 2 c) and mannose receptor (FIG. 2 d)expression are indicated as mean fluorescence intensity, whereas CD83 isindicated as the percentage of cells expressing this marker for maturedendritic cells (FIG. 2 b).

FIG. 3 shows induction of IL-12p70 secretion by monocyte derived DCafter stimulation with CD40 agonist antibodies and IFN-γ. Monocytederived immature DC were cultured for two days in the presence ofanti-CD40 monoclonal antibodies or isotype control antibodies alone orin combination with IFN-γ. Induction of IL-12p70 production requires thecombination of two different stimuli.

FIG. 4 shows IL-12p70 production induced by CD40 agonist antibodies andIFN-γ is blocked by pre-incubation with CD40-Fc. Pre-incubation of theCD40 agonist antibodies with an excess of CD40-Fc abolished the abilityof the anti-CD40 monoclonal antibodies to induce, in combination withIFN-γ, IL-12 production in monocyte derived DC.

FIG. 5 shows CD40 agonist monoclonal antibodies prime DC with anenhanced ability to induce CD8+ T cell responses. Monocyte derived DCwere either left unstimulated, or pre-activated with CD40 agonistantibody with or without IFN-γ and subsequently co-cultured withpurified autologous CD8+ T cells in the presence of a flu matrix peptiderepresenting a dominant HLA-A2 restricted epitope recognized by CD8+ Tcells. The induction of CD8+ T cell responses by CD40 activated DC wasstudied by analyzing both the expansion of flu peptide specific CD8+ Tcells 9 (FIG. 5 a) and the increase in CD8+ T cells that produce IFN-γ(FIG. 5 b).

DETAILED DESCRIPTION OF THE INVENTION

The molecules described and claimed include monoclonal antibodies,fragments and analogues thereof, peptides and other chemical entities.Monoclonal antibodies can be made by the conventional method ofimmunization of a mammal, followed by isolation of the B cell producingthe monoclonal antibodies of interest and fusion with a myeloma cell.The preferred monoclonal antibodies, and fragments and analoguesthereof, include chimeric antibodies, humanized antibodies, humanantibodies, Deiummunized™ antibodies, single-chain antibodies andfragments, including Fab, F(ab′)₂, Fv and other fragments which retainthe antigen binding function of the parent antibody. Single chainantibodies (“ScFV”) and the method of their construction are describedin U.S. Pat. No. 4,946,778.

Chimeric antibodies are produced by recombinant processes well known inthe art, and have an animal variable region and a human constant region.Humanized antibodies correspond more closely to the sequence of humanantibodies than do chimeric antibodies. In a humanized antibody, onlythe complementarity determining regions (CDRs), which are responsiblefor antigen binding and specificity, are non-human derived and have anamino acid sequence corresponding to the non-human antibody, andsubstantially all of the remaining portions of the molecule (except, insome cases, small portions of the framework regions within the variableregion) are human derived and have an amino acid sequence correspondingto a human antibody. See L. Riechmann et al., Nature; 332: 323-327 1988;U.S. Pat. No. 5,225,539 (Medical Research Council); U.S. Pat. Nos.5,585,089; 5,693,761; 5,693,762 (Protein Design Labs, Inc.).

Human antibodies can be made by several different methods, including byuse of human immunoglobulin expression libraries (Stratagene Corp., LaJolla, Calif.; Cambridge Antibody Technology Ltd., London, England) toproduce fragments of human antibodies (V_(H), V_(L), Fv, Fd, Fab, or(Fab′)₂), and use of these fragments to construct whole human antibodiesby fusion of the appropriate portion thereto, using techniques similarto those for producing chimeric antibodies. Human antibodies can also beproduced in transgenic mice with a human immunoglobulin genome. Suchmice are available from Abgenix, Inc., Fremont, Calif., and Medarex,Inc., Annandale, N.J. In addition to connecting the heavy and lightchain Fv regions to form a single chain peptide, Fab can be constructedand expressed by similar means (M. J. Evans et al., J. Immunol. Meth.,184: 123-138 1995).

DEIMMUNIZED™ antibodies are antibodies in which the potential T cellepitopes have been eliminated, as described in International PatentApplication PCT/GB98/01473. Therefore, immunogenicity in humans isexpected to be eliminated or substantially reduced when they are appliedin vivo.

All of the wholly and partially human antibodies described above areless immunogenic than wholly murine or non-human-derived antibodies, asare the fragments and single chain antibodies. All these molecules (orderivatives thereof) are therefore less likely to evoke an immune orallergic response. Consequently, they are better suited for in vivoadministration in humans than wholly non-human antibodies. especiallywhen repeated or long-term administration is necessary, as may be neededfor treatment of psoriasis or other inflammatory skin conditions.

Bispecific antibodies can be used as cross-linking agents between CD40of the same cell, or CD40 on two different cells. Such bispecificantibodies would have one specificity for each of two different epitopeson CD40. Bispecifics in which one specificity is a strong activator ofbinding of sCD40L to CD40, and one specificity is a partial ornon-inhibitor of binding of sCD40L to CD40, could synergize theagonistic effect on cross-linking.

These antibodies and the method of making them are described in U.S.Pat. No. 5,534,254 (Creative Biomolecules, Inc.). Different embodimentsof bispecific antibodies described in the patent include linking singlechain Fv with peptide couplers, including Ser-Cys, (GIY)₄-Cys,(His)₆-(Gly)₄-Cys, chelating agents, and chemical or disulfide couplingsincluding bismaleimidohexane and bismaleimidocaproyl.

Non-antibody molecules can be isolated or screened from compoundlibraries by conventional means. An automated system for generating andscreening a compound library is described in U.S. Pat. Nos. 5,901,069and 5,463,564. A more focused approach involves three-dimensionalmodeling of the binding site, and then making a family of moleculeswhich fit the model. These are then screened for those with optimalbinding characteristics.

Another approach is to generate recombinant peptide libraries, and thenscreen them for those which bind to the epitope of CD40 of interest.See, e.g., U.S. Pat. No. 5,723,322. This epitope is the same as thatbound by the monoclonal antibodies described in the examples below.Molecules can, in fact, be generated or isolated with relative ease inaccordance with techniques well known in the art once the epitope isknown.

Another approach is to induce endogenous production of the desiredanti-CD40 antibodies, by administering a peptide or an antibody whichinduces such production, or through gene therapy, where a gene encodingan appropriate anti-CD40 molecule or a fragment thereof is administered.The method of making and administering any of these molecules is wellknown in the art.

The molecules can be administered by any of a number of routes. In thecase of peptides and antibodies, because they are subject to degradationin the gastro-intestinal tract, they would preferably be injected. Othercompounds of the invention could also be injected. The injections couldbe intra-muscular, intra-venous or sub-cutaneous.

Non-peptide molecules of the invention could be administered orally,including by suspension, tablets and the like. Liquid formulations couldbe administered by inhalation of lyophilized or aerosolizedmicrocapsules. Suppositories could also be used.

Additional pharmaceutical vehicles could be used to control the durationof action of the molecules of the invention. They could be entrapped inmicrocapsules prepared by coacervation techniques or by interfacialpolymerization (hydroxymethylcellullose or gelatin microcapsules) incolloidal drug delivery systems (for example, liposomes, albuminmicrospheres, micro-emulsions, nanoparticles and nanocapsules) or inmacro-emulsions.

Excipients, for example, salts, various bulking agents, additionalbuffering agents, chelating agents, antioxidants, cosolvents and thelike can be included in the final formulation. Specific examples includetris-(hydroxymethyl) aminomethane salts (“Tris buffet”) and disodiumedetate.

The dosage and scheduling for the formulation which is selected can bedetermined by standard procedures, well known in the art. Suchprocedures involve extrapolating an estimated dosing schedule fromanimal models, and then determining the optimal dosage in a humanclinical dose ranging study.

Examples of molecules of the invention are set forth below.

Making and Using Agonistic Monoclonal Antibodies

In the Examples set forth below, the following procedures were used, asindicated in the examples.

Cell Lines and Culture Conditions

The EBV-transformed B-cell line JY and the myeloid derived cell lineTHP1 were cultured in T75 culture flasks routinely in lscove's modifiedDulbecco's medium (IMDM) to which 50 μg/ml gentamycin and 2% heatinactivated foetal calf serum was added (FCSi; BioWhittaker, Verviers,Belgium). The cells were cultured in a humidified incubator at 37° C.and 5% CO₂. Once or twice per week the cells were split ( 1/20 to1/100). To store the cell line, ampoules were made containing 5-10×10⁶cells/ml Hank's balanced salt solution HBSS supplemented with 20% FCSiand 10% DMSO, and stored in the liquid nitrogen.

Peripheral Mononuclear Blood Cell Isolation and Storage

Peripheral mononuclear blood cells (PBMC) were isolated from “buffycoats” of healthy donors by Lymphoprep™ (1.077 g/ml) densitycentrifugation and resuspended in Ca²⁺/Mg²⁺-free PBS-0.1% BSA.Autologous PBMC were stored in RPMI 1640 supplemented with 2 mML-glutamine, 10% FCSi, 50 μg/ml gentamycin and 10% DMSO at −196° C. (forCD8 T cell purification, see below).

Monocyte Enrichment and Generation of Monocyte-Derived Immature DC

Monocytes were purified from PBMC by immunomagnetic depletion(monocyte-enrichment cocktail containing Mabs against CD2, CD3, CD16,CD19, CD56, CD66b and glycophorin A; StemSep™ from StemCellTechnologies, Vancouver, Canada). Monocyte (>90% CD14⁺) preparationsdevoid of neutrophilic granulocytes, platelets, lymphocytes and NK cellswere subsequently cultured in serum-free culture medium, StemSpan™(StemCell Technologies), supplemented with 10 ng/ml GM-CSF and 20 ng/mlIL-4 (both cytokines from PeproTech, Rocky Hill, N.J., USA) at 37° C./5%CO₂ during 6-7 days. These monocytes were seeded at a cell density of1×10⁶/2 ml/10 cm² polystyrene surface (coated with 12 mg/ml/10 cm²poly-hydroxethylmethacrylate; Sigma) and fresh GM-CSF/IL-4 was added atday 2 and 5. After 6-7 days, the nonadherent cells (with a dendriticmorphology) were collected and displayed the following (flow cytometry,see below) phenotypic profile: CD1a⁺, CD14⁻, CD40⁺, C80⁺, CD83⁻, CD86⁺,HLA-DR⁺ and mannose receptor⁺⁺.

CD8 T Lymphocyte Isolation

Autologous PBMC were thawed, and CD8 T lymphocytes were purified byimmunomagnetic depletion of other cell types (CD8-enrichment cocktailwith Mabs against CD4, CD14, CD16, CD56 and glycophorin A; StemCellTechnologies). This procedure resulted in >90% CD3⁺/CD8⁺ lymphocytesdevoid of monocytes, neutrophilic granulocytes, platelets, B and CD4lymphocytes, and NK cells.

Flow Cytometric Analyses

Cells (0.1×10⁶ cells/100 μl PBS-0.1% BSA/sample) were incubated withconjugated (to either fluorescein isothiocyanate, phycoerythrin orperidinin chlorophyll protein) Mabs (Becton & Dickinson, Woerden, TheNetherlands) for 15 min. at 21° C., and then thoroughly washed inPBS-0.1% BSA and analyzed on a flow cytometer (FACSCalibur™; Becton &Dickinson, Woerden, The Netherlands).

Competition of CD40 Ligand and Anti-CD40 Monoclonal Antibodies on JYCells

Blocking of soluble CD40 ligand (sCD40L) binding by anti-CD40 monoclonalantibodies (Mabs) was demonstrated by using JY cells, which express highlevels of CD40. These cells (0.1×10⁶ cells/100 μl PBS-0.1% BSN/sample)were pre-incubated with anti-CD40 Mabs for 15 min. at 21°, and thenthoroughly washed in PBS-0.1% BSA, followed by an incubation with asoluble fusion protein consisting of the extracellular domain of humanCD40L fused to the extracellular domain of murine CD8α (CD40L-mCD8α;Kordia, Leiden, The Netherlands and Tanox Pharma BV, Amsterdam, TheNetherlands) for 15 min. at 21° C. Subsequently, CD40L-mCD8α wasdetected by using rat anti-mouseCD8α coupled to phycoerythrin, andanalyzed by flow cytometry.

Well-characterized control Mabs were included as controls: M2 and G28-5compete for the CD40L binding site, and 5C3 binds to a region distinctfrom the CD40L binding site. EA-5 partially inhibits the binding ofCD40L to its receptor (Pound et al., Int Immunol 1999, 11, p11-20).

Inhibition of Binding of Anti-CD40 Monoclonal Antibodies to MembraneCD40L by CD40-Fc (IgG4).

As a source of membrane CD40L activated CD4+ T cells are used. To thispurpose expression of CD40L on T cells is induced through culturingplastic non-adherent PBMC with PMA and ionomycine for 6 hrs in IMDM+5%human pooled AB serum. CD40-Fc (IgG4 made by Tanox Inc Houston USA) isdirectly added at a saturating dose to the activated T cells or afterpre-incubation of CD40-Fc with excess of anti-CD40 Mabs. Binding ofCD40-Fc to CD40L activated CD4+ (CD3+CD8−) T cells is monitored throughFACS analysis after staining with PE conjugated goat anti human IgG-Fc,FITC conjugated CD3 and PERCP conjugated CD8.

CD40 ELISA

ELISA plates (Immunon 2) were coated overnight at room temperature with0.5 μg/ml, 50 μl per well of goat-anti-human IgG (Fc). Next the plateswere treated with 1% BLOTTO for 60 min at room temperature. Afterwashing 4 times with PBS/Tween, 50 μl/well of CD40-Fc plus 50 μl ofsupernatants of the fusion wells were added and incubated for 1 hour atroom temperature. After 4 washings with PBS/Tween, 50 μl ofgoat-anti-mouse IgG (Fc)-HRP conjugate was added and incubated for 1hour. After 4 more washings the substrate TMB was added at 100 μl/wellto the plates, which were then incubated for 30 min. The reaction wasstopped by addition of 50 μl/well of 0.2 M H₂SO₄ and the plates wereread with an ELISA reader at 450/590 nm.

THP-1 Assay

Stimulation of THP-1 Cells

3×10⁶ THP-1 cells were first cultured for two days in 10 ml of IMDM+2%of human type AB serum in the presence of 5×10² U/ml IFN-γ. Next theIFN-γ treated THP-1 cells were washed once in IMDM+2% human type ABserum. 10⁴ THP-1 cells per 96 well plate well were cultured for two daysin 120 μl of culture medium diluted 1:2 with hybridoma supernatant. Ascontrols CD154-mCD8 was used at 40 μg/ml maximum and 2× dilutions andLPS at 20 ng/ml maximum and 2× dilutions.

Measurement of IL-8

ELISA plates were coated with mouse anti human IL-8 antibody (Serotec)at 5 μg/ml, 100 μl/well for 2 hrs at room temperature on a plate shaker.The plates were then incubated with 1% BLOTTO for one hour on the plateshaker at room temperature. After four washings with PBS/Tween, 80 μl ofsupernatants harvested from the THP-1 plate were added to the ELISAplate. For the IL-8 standards: IL-8 was diluted with 1% BLOTTO to 1000pg/ml, 300 pg/ml, 100 g/ml, 30 pg/ml, 10 pg/ml, 3 pg/ml,1 pg/ml. TheELISA plates were incubated for one hr at room temperature on the plateshaker. After four washings with PBS/Tween, 100 μl/well mouse-anti IL-8biotin conjugate (Serotec) was added at 1:1000 dilution in 1% BLOTTO andthe plates were incubated for one hour at room temperature. After fourwashings with PBS/Tween, 100 μl/well AMDX SA-HRP at 1:1000 dilution in1% BLOTTO was added to the wells and the plates were incubated for 1hour at room temperature on the plate shaker. After 4 washings withPBS/Tween, 100 μl of TMB substrate was added to each well and the plateswere incubated for 30 minutes at room temperature on the plate shaker.The reaction was stopped by addition of 50 μl/well of 0.2 M H₂SO₄ andthe plates were read with an ELISA reader at 450/590 nm.

Induction of Mature DC

Immature DC (see above) are cultured in the presence of anti-CD40 Mabsunder serum-free condition (StemSpan™) at 37° C./5% CO₂ for 48 hours. Inaddition, CD40L-mCD8α, LPS and a combination of IL-1β and TNF-α are usedas well-established controls for DC maturation. The change from immatureto mature DC is determined by: (1) phenotype (CD1a⁺, CD14⁻, CD40⁺⁺⁺,CD80⁺, CD83⁺, CD86⁺⁺⁺, HLA-DR⁺⁺⁺, mannose receptor⁻), (2) IL-12p70production (commercially available kit), and by (3) the capability ofinducing influenza-matrix peptide specific autologous cytotoxic CD8⁺ Tlymphocytes (see below).

IL-12p70 ELISA

Immature DC are cultured in the presence of anti-CD40 Mabs (1 μg/ml)with or without IFN-γ (1000 U/ml) for 48 hrs. IL-12p70 secretion wasmeasured in the supernatant using a commercially available kit fromDiaclone Research, Becanson, France. Inhibition of IL-12 production wasobtained by preincubation of anti-CD40 Mabs with 10 times excess ofCD40-Fc (IgG4 made by Tanox Inc Houston USA) for 15 min at roomtemperature.

Induction of Cytotoxic CD8⁺ T Lymphocytes by Mature DC

Mature DC generated by agonistic anti-CD40 Mabs are loaded with asynthetic influenza matrix peptide (Flu-peptide 58-66); 1×10⁶DC/Flu-peptide 5 μg/ml StemSpan™) and co-cultured with 0.5×10⁶ purifiedautologous CD8+ T lymphocytes at 37° C/5% CO₂ during 7 days.Cytotoxicity of the CD8+ T lymphocytes is determined by: (1) enumerationof the number of IFN-γ producing T cells, which are representative foractivated CTL (using flow cytometry with an IFN-γ detection kit fromMiltenyi Biotec, Bergisch Gladbach, Germany), and (2) a conventionalassay measuring cytolysis by CTL of target cells loaded withFlu-peptide.

EXAMPLES Example 1

Generation of Mouse Anti-Human CD40 Monoclonal Antibodies

Two immunization protocols were used to generate anti-CD40 monoclonalantibodies. In the first, female BALB/c mice were injectedintraperitoneally with SF-9 cells expressing CD40 (3×10⁶ cells/mouse),which were washed with PBS twice before injection. At day 17 and 31 themice received a booster injection with SF-9 cells. Fourteen days afterthe last the last the injection the spleen cells were isolated and 10⁸cells were used for cell fusion with 10⁸ SP2/0 murine myeloma cellsusing polyethylene glycol. The fused cells were resuspended in D15 (amodified DMEM medium) supplemented with HAT, followed by plating onfifty-one 96 wells plates. After 10-14 days supernatants of wellscontaining growing hybridoma cells were screened for anti-CD40 antibodyproduction in an ELISA. This analysis showed that a total of 69 wellsout of 4896 seeded wells contained hybridomas producing anti-human CD40specific antibodies. Culture supernatants of these wells were selectedfor additional experiments, such as for studying the induction of IL-8secretion from THP-1 cells (see below). Next limiting dilution wasperformed twice to obtain clones from a number of hybridoma lines thatproduced CD40 agonist antibodies. For this purpose hybridoma cells wereseeded at densities of less than 1 c/well in 96 well plates and culturedfor 3-4 weeks. Supernatant of positive wells was screened in the CD40ELISA and the THP-1 assay for the presence of CD40 binding antibodies.

For the second immunization regimen, BALB/c mice were injectedintraperitoneally with 2.5×10⁶ monocyte-derived immature DC. At days 14,35 and 55 mice received booster injections with monocyte-derived DC fromdifferent donors. At around day 100-120, spleen cells will be isolatedand fused with murine myeloma cells in analogy to the above protocol.Supernatants of wells with growing hybridomas will be screened for thepresence of CD40 binding antibodies in the ELISA. Hybridoma supernatantscontaining CD40 binding antibodies will be subsequently screened forpotential agonistic activity as described for the hybridoma'soriginating from B cells isolated from the BALB/c immunized with CD40expressing SF-9 cells.

Example 2

Screening the CD40 Binding Antibody Samples from Hybridoma Lines forAgonistic Activity on THP-1 Cells and Subsequent Cloning of Lines andTesting of Monoclonal Mabs

To screen for antibodies with agonistic activity, the selectedsupernatants containing CD40 binding antibodies were subsequently testedfor their ability to induce IL-8 production in the CD40 expressingmonocytic cell line THP-1, which had been pre-incubated with IFN-γ. Mostsupernatants contained anti-CD40 antibodies, which displayed agonisticactivity in this assay. Supernatants were arbitrarily subdivided intofour different groups on the basis of their performance in the THP-1assay (strong agonists with an OD of >2.000, intermediate agonists withan OD between 1.000-2.000, low agonists with an OD between 0.375-0.999and non-agonists with an OD <0.375).

A number of the hybridoma lines were cloned and monoclonal antibodiesfrom the resulting clones were also tested in the THP-1 assay. Most butnot all clones retained the reactivity pattern of the correspondingmother lines (data not shown).

Example 3

Assaying the Ability of the CD40 Reactive Antibody Clones to DriveMaturation. IL-12p70 Production and Priming for CTL Activation ofImmature DC

DC derived from monocytes after culture with GM-CSF and IL-4 representimmature DC. Anti-CD40 monoclonal antibodies have been assayed for theircapacity to induce maturation of these CD40 expressing immature DC.Experiments from other investigators have shown that stimulation ofmonocyte-derived DC with sCD40L results in their differentiation into DCwith a mature phenotype. Furthermore, sCD40L in combination with IFN-γstimulates monocyte-derived DC to secrete IL-12p70. In contrast toimmature DC, mature DC express CD83. In addition, compared to immatureDC, mature DC display enhanced expression on a per cell basis of theco-stimulatory molecules CD80 and CD86, decreased expression of themannose receptor and loss of the ability to efficiently take upmolecules, as shown for dextran-FITC. At first the phenotypical changesthat accompany the differentiation of immature to mature DC weremonitored by FACS-analysis as a read-out for induction of DC maturationby the anti-CD40 monoclonal antibodies. Antibodies were first used ontheir own to stimulate monocyte-derived DC. As shown in FIG. 1 (combinedresults of several experiments showing CD83 up-regulation and mannosereceptor down-regulation) and in FIG. 2 (one typical experiment showinginduced expression of CD80, CD83 and CD86) CD40 binding antibodies weretested and were found to induce phenotypical maturation of monocytederived DC, as is indicated by the increased percentage of cellsexpressing the CD83 marker, the increased expression on a per cell basis(mean fluorescence intensity; MFI) of CD80 and CD86 and decreasedexpression of the mannose receptor. Remarkably, some of the clones thatdid not induce IL-8 production in THP-1 cells could induce maturation ofDC, demonstrating that agonist properties of CD40 monoclonal antibodiesmay differ between different CD40 expressing cell types (data notshown).

In addition, the IL-12p70 production of monocyte derived DC was testedafter stimulation with the CD40 monoclonal antibodies and IFN-γ, sincedendritic cells require stimulation through at least two differentpathways to produce IL-12p70 (Kalinski et al Blood 1997 90:1926). Ourresults show that apart from induction of phenotypical maturation, theCD40 agonist antibodies also induced IL-12 production in DC when usedtogether with IFN-γ (FIG. 3). Our finding that pre-incubation of theCD40 monoclonal antibodies with excess of CD40-Fc inhibited induction ofIL-12 production demonstrated that the agonistic effect of theantibodies is really exerted through CD40 and not through other membraneexpressed molecules on the DC (FIG. 4).

In the mouse, T cell help to CTL was found to be mediated through CD40activated DC. Antigen dependent interaction of helper T cells with DCdid not only result in the activation of the helper T cell, but throughCD40L-CD40 interaction also in the activation of the DC. Only in theiractivated stage DC were able to prime CTL responses. In the absence ofhelper T cells no DC activation and therefore no CTL priming occurred.However, by means of in vivo administration of an anti-mouse CD40stimulatory antibody, T cell help could be efficiently bypassed and DCdirectly activated.

To show that the same mechanism of CTL activation applies to man, an invitro study was performed in which CTL activation was studied in aco-culture system consisting of purified human CD8⁺ T cells,monocyte-derived DC as APC and a minimal peptide derived from influenzavirus matrix protein as antigen. This peptide constitutes a dominantHLA-A2 restricted CTL epitope. This experiment was carried out toestablish whether our anti-CD40 monoclonal antibodies could empowermonocyte-derived DC with an increased ability to stimulate CTL responsescompared to untreated control DC. CTL activation was analyzed in thisexperiment through measurement of production of IFN-γ by activated CTLand enumeration of expansion of CTL with PE conjugated HLA-A2/influenzamatrix peptide tetramers. As shown in FIGS. 5 a and b the stimulation ofmonocyte derived DC with CD40 monoclonal antibodies led to increasedability of these cells to induce a flu peptide directed CD8⁺ T cellresponse. For most antibodies this effect was elevated when, in additionto the monoclonal antibodies, IFN-γ was used in the pre-activation ofthe dendritic cells.

Example 4

Analysis of the Inhibition of the Binding of sCD40L to CD40 by theAnti-CD40 Antibody Samples

Anti-CD40 antibodies that synergize with sCD40L in the induction of CD40mediated activation of DC most likely show co-binding with sCD40L toCD40 and thus do not display strong blocking of binding of sCD40L toCD40. To screen for such antibodies, the percentage of inhibition ofsCD40L binding to CD40 on JY EBV transformed B cells by the monoclonalantibodies was tested. This analysis revealed that there was strongvariation in the degree that the monoclonal antibodies could inhibit thebinding of sCD40L to CD40. Some antibody samples almost completelyinhibited sCD40L binding, whereas other antibody samples could onlypartially block sCD40L binding or had no effect at all (table 2). Theresults were confirmed in the reverse way for a limited number of clonesby testing the inhibition caused by the anti-CD40 monoclonal antibodiesof the binding of CD40-Fc to CD40L expressed on the membrane ofPMA+ionomycine activated CD4+ T cells. In this experiment clone 4blocked binding of CD40-Fc to CD40L on the T cells for 88%, clone 7 and64 for respectively 16% and 25%. Although there was no absolutecorrelation between the performance of the antibodies in the DCmaturation and the THP-1 assay and their ability to block sCD40L bindingto CD40, all the clones that did not block this interaction werenon-responders in both assays (data not shown)

Example 5

Synergism Between Anti-CD40 Antibodies and mCD40L or sCD40L in AgonistActivity on DC

It is predicted that those antibodies that to a major extent blockbinding of sCD40L to CD40 will not display synergism with sCD40L in theinduction of DC maturation or other agonistic properties exerted on CD40positive cells. In contrast, some of the CD40 binding antibodies thatefficiently co-bind with sCD40L to its receptor will presumably showsynergism with sCD40L or membrane bound CD40L (mCD40L) in driving DCmaturation (as a source of membrane bound CD40L antigen or mitogenactivated CD4+ T cells will be used). This will be demonstrated by theincreased percentage of cells expressing CD83, by the increasedexpression on a per cell basis of CD80 and CD86 and the decreasedexpression of the mannose receptor. Also the level of IL-12p70 producedby the DC after stimulation by the combination of one of theseantibodies with sCD40L and IFN-γ will be enhanced compared to the levelinduced by sCD40L and IFN-γ alone. Apart from synergism between sCD40Land an anti-CD40 antibody, two anti-CD40 antibodies may also showsynergism with each other in the induction of IL-12p70 secretion. Thissynergism may occur most noticeably between antibodies that blockbinding of sCD40L to CD40 and those that are partial or non-inhibitorsof this interaction, as these antibodies are expected to bind differentepitopes on CD40.

In analogy to the experiment in which the maturation of DC was tested,the effect on CTL activation of the anti-CD40 antibody samples, used ontheir own or together with sCD40L, will be evaluated in futureexperiments. It is expected that, resulting from more efficientstimulation of the DC, synergism in CTL activation will occur betweenthe same combinations of sCD40L and monoclonal antibodies as in thematuration assay. The same holds true with regard to synergism in CTLactivation between two different anti-CD40 antibodies.

Example 6

Enhanced Potency, in Comparison to CD40 Agonist Antibody, of aBispecific Antibody Directed to CD40 and 4-1 BB Ligand or a BispecificAntibody Directed to CD40 and CD28 in the Ability to License DC for CTLActivation

Use of a bi-specific antibody with specificity for CD40 on one side anda determinant on T cells on the other side potentially has the benefitof bringing the activated DC in close contact with surrounding T cells.If the antibody part that recognizes the T cell determinant hasagonistic properties, the additional benefit may be that the attracted Tcell will be stimulated both through the signals delivered by theactivated DC and the agonistic properties of the T cell part of thebispecific antibody. This possibility will be evaluated by comparing theeffect of the addition of the CD40 monoclonal antibodies and thebi-specific antibodies in the above described DC-CTL co-culture system,using flu peptide specific CD8+ T cell responses as read out.

The description and examples are exemplarily only and not limiting, andthe invention is defined only by the claims which follow, and includesall equivalents, known and unknown, of such claimed subject matter.

Various modifications of the invention in addition to those describedherein will become apparent to those skilled in the art from theforegoing description and the accompanying figures. Such modificationsare intended to fall within the scope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1. An anti-CD40 monoclonal antibody, wherein said anti-CD40 antibody isproduced by a hybridoma selected from the group consisting of hybridomasdeposited with American Type Culture Collection (ATCC) as depositdesignation PTA-2993, PTA-2994, PTA-2995, PTA-2996, PTA-2997, PTA-2998and PTA-2999.
 2. A hybridoma selected from the group consisting ofhybridomas deposited with American Type Culture Collection (ATCC) asdeposit designation PTA-2993, PTA-2994, PTA-2995, PTA-2996, PTA-2997,PTA-2998 and PTA-2999.
 3. A composition comprising a combination of twoor more monoclonal antibodies according to claim
 1. 4. A cell line thatproduces the anti-CD40 monoclonal antibody of claim
 1. 5. An anti-CD40monoclonal antibody that is a chimeric antibody, humanized antibody,single chain antibody or CD40-binding fragment thereof, of the antibodyaccording to claim
 1. 6. The anti-CD40 monoclonal antibody according toclaim 5 from which the potential human T cell epitopes have beeneliminated.
 7. The CD40-binding fragment according to claim 5, which isan Fab, F(ab′)₂, Fv, or single chain Fv fragment.
 8. A compositioncomprising a combination of two or more chimeric antibodies, humanizedantibodies, single chain antibodies or CD40-binding fragments thereofaccording to claim
 5. 9. A composition comprising a combination of oneor more anti-CD40 monoclonal antibodies according to claim l with one ormore chimeric antibodies, humanized antibodies, single chain antibodiesor CD40-binding fragments thereof according to claim
 5. 10. A cell linethat produces the chimeric antibody, humanized antibody, single chainantibody or CD40-binding fragment thereof according to claim 5.